In situ hybridization (ISH) is a technique that allows detection and localization of specific nucleic acid molecules in morphologically preserved individual cells, histological tissue sections, or chromosome preparations. It was first described in 1969 and is based on the complementary hybridization of a nucleotide probe to a specific target sequence of DNA or RNA in the cell. This may be endogenous DNA, messenger RNA (mRNA), micro RNA (miRNA), viral sequences, or bacterial sequences. The added probe is labeled with a reporter molecule and sites of binding are visualized either fluorescently (Fluorescent in sit hybridization, FISH) or chromogenically (Chromogenic in situ hybridization, CISH).
After completion of sequencing the whole human genome, many thousands of novel human gene sequences have been annotated in public and private databases in recent years. This opened a new door for ISH, since it is relatively easy, fast and inexpensive to design and synthesizes antisense probes for detecting a specific sequence of any novel gene in the cell. The tissue-specific and cell-type specific express patterns together with expression level of the gene can be obtained with ISH, which will provide valuable information for analyzing the function of the gene.
However, the application of ISH techniques can be limited by their inability to detect DNA or RNA targets with low copy numbers in cells due to lack of sensitivity and specificity. It is especially challenging to apply ISH into the clinical setting where formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide to store clinical tissue samples. The FFPE method preserves tissue morphology well but the cross-linking fixation by formaldehyde can cause poor accessibility of the target sequence to the detection probe, chemical or physical interaction of the probe molecules with other molecules or structures, and damage to nuclear acids, especially mRNAs.
In order to circumvent the limitation of ISH and extend its utility in diagnostic pathology, several strategies have been developed to improve the sensitivity of ISH. Recently, a new ISH signal amplification method called RNAscope® was developed by Advanced Cell Diagnostics. Inc. (U.S. Pat. No. 7,709,198). This assay includes uniquely designed oligo capture probes and a signal amplification system composed of preamplifiers, amplifiers, and label probes, allowing substantial signal amplification without amplifying background signal, enabling single RNA molecule detection for virtually any genes. An exemplary embodiment of the RNAscope® technology is schematically illustrated in FIG. 1 and will be described in detail in Section 2 of this application.
In a typical RNAscope® assay for detecting target nucleic acid, a target mRNA whose expression is to be detected is released from cells and captured on a solid surface (e.g., a well of a microtiter plate). A set of two or more capture probes and a signal generating multimer are also provided. The capture probes hybridize to both target nucleic acid and signal generating multimer and thus capture signal generating multimer to target nucleic acid. The signal generating multimer comprises label probes (LPs). But more typically, the signal generating multimer comprises preamplifiers and/or amplifiers in addition to label probes. The label probe is capable of binding to a label particle or molecule that provide detectable signal. The label probe has a larger molecular structure enabling that attachment of a plurality of label particle or molecules that provide stronger signal than a single label particle or molecule. Thus, RNAscope® improves the sensitivity and specific of nucleic acid detection.
However, RNAscope® alone still cannot reliably detect some low copy genes in retrospective Formalin-Fixed. Paraffin-Embedded (FFPE) tissue sections where RNA is significantly degraded. Also, single RNA molecule cannot to be visualized at 40× magnification using current RNAscope® technology. It is desirable to further enhance the detection signal to enable more robust detection of any RNA molecules, including the significantly degraded RNA molecules, and to allow easy visualization of detected RNA signal at 10× magnification.
Here we describe a novel ISH signal amplification assay to extend the utility of ISH in diagnostic pathology by combining two signal amplification methods into one system. This system has three formats: (1) RNAscope® in combination with biotin-(strept)avidin, (2) RNAscope® in combination with an antibody, (3) RNAscope® in combination with Tyramide Signal Amplification (TSA). All these methods can leverage the exceptional specificity of RNAscope® technology with the amplification power of the three amplification methods. We surprisingly found that the above three combined signal amplification methods can highly amplify signals associated with the presence of a target nucleic acid in sample while having excellent signal to noise ratio.
Biotin-avidin (or biotin-streptavidin) is a well known signal amplification system base on the facts that the two molecules have extraordinarily high affinity to each other and that one avidin/streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. TSA is based on the deposition of a large number of haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. In the presence of small amounts of hydrogen peroxide, immobilized Horse Radish Peroxidase (HRP) converts the labeled substrate into a short-lived, extremely reactive intermediate. The activated substrate molecules then very rapidly react with and covalently bind to electron-rich moieties of proteins, such as tyrosine, at or near the site of the peroxidase binding site. In this way, a lot of extra hapten molecules conjugated to tyramide can be introduced at the hybridization site in situ. Subsequently, the deposited tyramide-hapten molecules can be visualized directly or indirectly.
All of these signal amplification methods have been used in ISH with different degrees of success but usually the signal intensity and/or signal to noise ratio are not good enough for routine diagnostic pathology. In the case of TSA, in particular, when used alone in ISH, produces a high degree of background noise. The day-to-day and laboratory-to-laboratory consistency is also less than desirable.
In view of the above, a need exists for methods of amplifying nucleic acids with both high intensity and high specificity. A need also exists for such a method with strong consistency. The present invention provides these and other features that will be apparent upon review of the following.